Chaperone proteins in the heat shock protein-70 (hsp70) family possess endogenous ATP-binding, ATPase activity and interact with intracellular protein substrates in an ATP-dependent manner; the hydrolysis of ATP to ADP results in an increase in the affinity of the chaperone for protein substrates. Hsp70s can also specifically interact with 25-hydroxylated vitamin D metabolites. Using constitutively-expressed heat shock protein-70 (hsc70) as chaperone, here we demonstrate that vitamin D metabolite binding to hsc70 is also ATP-dependent. Transient over-expression of an hsc70-GFP chimeric construct in primate kidney cells resulted in a 6-fold increase in specific, extractable 25-hydroxyvitamin D₃ (25OHD₃) binding. When ATPase capability of hsc70 was disabled, this increase was completely blocked. In solution, the binding of 25-hydroxylated vitamin D metabolites to hsc70 was significantly increased (P < 0.01) in the presence of ATP and a non-metabolizable ATP analog. The ATP-directed increase in specific binding resulted from an increase in the abundance of relatively "high affinity" hormone-binding sites (K_d0.24 nM). These results suggest that ATP hydrolysis to ADP would favor the release of vitamin Ds from a donor hsc70 molecule at a time when an hsc70- bound acceptor protein substrate is anchored to the chaperone with relative avidity. We theorize that the endogenous ATPase activity of hsc70 promotes the transfer of vitamin D sterols to other intracellular vitamin D binding proteins, such as the vitamin D receptor and vitamin D-hydroxylases, to which hsc70 is known to bind.